Methods and apparatus for detecting variants utilizing base stacking

ABSTRACT

Methods and apparatus are provided for the analysis and determination of the nature of repeat units in a genetic target. In one method of this invention, the nature of the repeat units in the genetic target is determined by the steps of providing a plurality of hybridization complex assays arrayed on a plurality of test sites, where the hybridization complex assay includes at least a nucleic acid target containing a simple repetitive DNA sequence, a capture probe having a first unique flanking sequence and n repeat units, where n=0,1,2 . . . , or fractions thereof, being complementary to the target sequence, and a reporter probe having a selected sequence complementary to the same target sequence strand wherein the selected sequence of the reporter includes a second unique flanking sequence and m repeat units, where m=0,1,2 . . . , or fractions thereof, but where the sum of repeat units in the capture probe plus reporter probe is greater than 0 (n+m&gt;0). Concordance and discordance among the hybridization complex assays at the test sites is determined at least in part by hybridization stability. Electronic stringency control may be utilized. Applications include paternity testing, forensic use, and disease diagnostics, such as for the identification of the existence of a clonal tumor.

RELATED APPLICATION INFORMATION

[0001] This application is a continuation of U.S. Ser. No. 09/645,757,filed Aug. 24, 2000, entitled “METHODS AND APPARATUS FOR DETERMINATIONOF LENGTH POLYMORPHISMS IN DNA”, now allowed, which is a continuation ofU.S. Ser. No. 09/030,156, filed Feb. 25, 1998, entitled “METHODS ANDAPPARATUS FOR DETERMINATION OF LENGTH POLYMORPHISMS IN DNA”, now issuedas U.S. Pat. No. 6,207,363.

[0002] Further, this application is related to Application Ser. No.08/986,065, filed Dec. 5, 1997, entitled “METHODS AND PARAMETERS FORELECTRONIC BIOLOGICAL DEVICES”, now issued as U.S. Pat. No. 6,051,380,which is a continuation-in-part of application Ser. No. 08/534,454,filed Sep. 27, 1995, entitled “APPARATUS AND METHODS FOR ACTIVEPROGRAMMABLE MATRIX DEVICES”, now issued as U.S. Pat. No. 5,849,486,which is a continuation-in-part of application Ser. No. 08/304,657,filed Sep. 9, 1994, entitled “AUTOMATED MOLECULAR BIOLOGICAL DIAGNOSTICSYSTEM,” now issued as U.S. Pat. No. 5,632,957, which is acontinuation-in-part of application Ser. No. 08/271,882, filed Jul. 7,1994, entitled “METHODS FOR ELECTRONIC STRINGENCY CONTROL FOR MOLECULARBIOLOGICAL ANALYSIS AND DIAGNOSTICS,” now aissued as U.S. Pat. No.6,017,696, which is a continuation-in-part of Ser. No. 08/146,504, filedNov. 1, 1993, entitled “ACTIVE PROGRAMMABLE ELECTRONIC DEVICES FORMOLECULAR BIOLOGICAL ANALYSIS AND DIAGNOSTICS”, now issued as U.S. Pat.No. 5,605,662, all incorporated herein by reference as if fully setforth herein.

FIELD OF THE INVENTION

[0003] The methods and apparatus of these inventions relate to systemsfor genetic identification for disease state identification. Moreparticularly, the methods and apparatus relate to systems for thedetection of repeat unit states, such as the number of short tandemrepeat units for the identification of individuals such as in a forensicor paternity sense, or for determination of disease states, such as forclonal tumor detection.

BACKGROUND OF THE INVENTION

[0004] Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein. Many of these techniques andprocedures form the basis of clinical diagnostic assays and tests. Thesetechniques include nucleic acid hybridization analysis, restrictionenzyme analysis, genetic sequence analysis, and the separation andpurification of nucleic acids and proteins (See, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y,1989).

[0005] Most of these techniques involve carrying out numerous operations(e.g., pipetting, centrifugation, electrophoresis)on a large number ofsamples. They are often complex and time consuming, and generallyrequire a high degree of accuracy. Many a technique is limited in itsapplication by a lack of sensitivity, specificity, or reproducibility.For example, these problems have limited many diagnostic applications ofnucleic acid hybridization analysis.

[0006] The complete process for carrying out a DNA hybridizationanalysis for a genetic or infectious disease is very involved. Broadlyspeaking, the complete process may be divided into a number of steps andsubsteps. In the case of genetic disease diagnosis, the first stepinvolves obtaining the sample (blood or tissue). Depending on the typeof sample, various pre-treatments would be carried out. The second stepinvolves disrupting or lysing the cells, which then release the crudeDNA material along with other cellular constituents. Generally, severalsub-steps are necessary to remove cell debris and to purify further thecrude DNA. At this point several options exist for further processingand analysis. One option involves denaturing the purified sample DNA andcarrying out a direct hybridization analysis in one of many formats (dotblot, microbead, microplate, etc.). A second option, called Southernblot hybridization, involves cleaving the DNA with restriction enzymes,separating the DNA fragments on an electrophoretic gel, blotting to amembrane filter, and then hybridizing the blot with specific DNA probesequences. This procedure effectively reduces the complexity of thegenomic DNA sample, and thereby helps to improve the hybridizationspecificity and sensitivity. Unfortunately, this procedure is long andarduous. A third option is to carry out an amplification procedure suchas polymerase chain reaction (PCR), strand displacement amplification orother method. These procedures amplify (increase) the number of targetDNA sequences relative to non-target sequences. Amplification of targetDNA helps to overcome problems related to complexity and sensitivity ingenomic DNA analysis. After these sample preparation and DNA processingsteps, the actual hybridization reaction is performed. Finally,detection and data analysis convert the hybridization event into ananalytical result.

[0007] Nucleic acid hybridization analysis generally involves thedetection of a very small number of specific target nucleic acids (DNAor RNA) with an excess of probe DNA, among a relatively large amount ofcomplex non-target nucleic acids. The substeps of DNA complexityreduction in sample preparation have been utilized to help detect lowcopy numbers (i.e. 10,000 to 100,000) of nucleic acid targets. DNAcomplexity is overcome to some degree by amplification of target nucleicacid sequences using polymerase chain reaction (PCR) and other methods.(See, M. A. Innis et al, PCR Protocols: A Guide to Methods andApplications, Academic Press, 1990, Spargo et al., 1996, Molecular &Cellular Probes, in regard to SDA amplification). Amplification resultsin an enormous number of target nucleic acid sequences that improves thesubsequent direct probe hybridization step.

[0008] The actual hybridization reaction represents one of the mostimportant and central steps in the whole process. The hybridization stepinvolves placing the prepared DNA sample in contact with a specificreporter probe, at a set of optimal conditions for hybridization tooccur to the target DNA sequence. Hybridization may be performed in anyone of a number of formats. For example, multiple sample nucleic acidhybridization analysis has been conducted on a variety of filter andsolid support formats (See G. A. Beltz et al., in Methods in Enzymology,Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press,New York, Chapter 19, pp. 266-308, 1985). One format, the so-called “dotblot” hybridization, involves the non-covalent attachment of target DNAsto filter, which are subsequently hybridized with a radioisotope labeledprobe(s). “Dot blot” hybridization gained wide-spread use, and manyversions were developed (see M. L. M. Anderson and B. D. Young, inNucleic Acid Hybridization —A Practical Approach, B. D. Hames and S. J.Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985).It has been developed for multiple analysis of genomic mutations (D.Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for thedetection of overlapping clones and the construction of genomic maps (G.A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).

[0009] New techniques are being developed for carrying out multiplesample nucleic acid hybridization analysis on micro-formatted multiplexor matrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). Thesemethods usually attach specific DNA sequences to very small specificareas of a solid support, such as micro-wells of a DNA chip. Thesehybridization formats are micro-scale versions of the conventional “dotblot” and “sandwich” hybridization systems.

[0010] The micro-formatted hybridization can be used to carry out“sequencing by hybridization”(SBH) (see M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makesuse of all possible n-nucleotide oligomers (n-mers) to identify n-mersin an unknown DNA sample, which are subsequently aligned by algorithmanalysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov,Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4Genomics, 114, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA10089, 1992; and R. Drmanac and R. B. Crkvenjakov, U.S. Pat. No.5,202,231, Apr. 13, 1993).

[0011] There are two formats for carrying out SBH. The first formatinvolves creating an array of all possible n-mers on a support, which isthen hybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

[0012] Southern, United Kingdom Patent Application GB 8810400, 1988; E.M. Southern et al., 13 Genomics 1008, 1992, proposed using the firstformat to analyze or sequence DNA. Southern identified a known singlepoint mutation using PCR amplified genomic DNA. Southern also describeda method for synthesizing an array of oligonucleotides on a solidsupport for SBH. However, Southern did not address how to achieveoptimal stringency condition for each oligonucleotide on an array.

[0013] Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, usedthe second format to sequence several short (116 bp) DNA sequences.Target DNAs were attached to membrane supports (“dot blot” format). Eachfilter was sequentially hybridized with 272 labeled 10-mer and 11-meroligonucleotides. A wide range of stringency condition was used toachieve specific hybridization for each n-mer probe; washing timesvaried from 5 minutes to overnight, and temperatures from 0%C. to 16%C.Most probes required 3 hours of washing at 16%C. The filters had to beexposed for 2 to 18 hours in order to detect hybridization signals. Theoverall false positive hybridization rate was 5% in spite of the simpletarget sequences, the reduced set of oligomer probes, and the use of themost stringent conditions available.

[0014] A variety of methods exist for detection and analysis of thehybridization events. Depending on the reporter group (fluorophore,enzyme, radioisotope, etc.) used to label the DNA probe, detection andanalysis are carried out fluorimetrically, colorimetrically, or byautoradiography. By observing and measuring emitted radiation, such asfluorescent radiation or particle emission, information may be obtainedabout the hybridization events. Even when detection methods have veryhigh intrinsic sensitivity, detection of hybridization events isdifficult because of the background presence of non-specifically boundmaterials. A number of other factors also reduce the sensitivity andselectivity of DNA hybridization assays.

[0015] One form of genetic analysis consists of determining the natureof relatively short repeating sequences within a gene sequence. Shorttandem repeats (STR's) have been identified as a useful tool in bothforensics and in other areas (paternity testing, tumor detection, D.Sidransky, genetic disease, animal breeding). Indeed, the United StatesFederal Bureau of Investigation has announced that it is considering theuse of short tandem repeat sequences for forensic purposes. (Dr. BruceBudowle, DNA Forensics, Science, Evidence and Future Prospects, McLean,Va. November, 1997).

[0016] Various proposals have been made for identifying, amplifying,detecting and using polymorphic repeat sequences. For example, Tautz PCTW090/04040-PCT/EP98/01203, in an application entitled “Process for theAnalysis of Length Polymorphorisms in DNA Regions” (translated fromGerman), discloses a process for the analysis of length polymorphisms inregions of simple or cryptically simple DNA sequences. Tautz discloses amethod which includes these steps of addition of at least one primerpair onto the DNA that is to be analyzed, wherein one of the moleculesof the primer pair is substantially complementary to the complementarystrands of the 5′ respectively 3′ flank of a simple or crypticallysimple DNA sequence and wherein the addition takes place withinorientation that is such that the synthesis products obtained from aprimer controlled polymerization reaction with one of the two primerscan be used, following denaturation, as matrices for the addition of theother primer, performing a primer-controlled polymerization reaction andseparating, such as by normal gel electrophoresis the products andanalyzing the polymerase chain reaction products.

[0017] Caskey et al. at the Baylor College of Medicine also detectedpolymorphisms in a short tandem repeat by performing DNA profilingassays. In Caskey et al., U.S. Pat. No. 5,364,759, issued Nov. 15, 1994,entitled “DNA Typing With Short Tandem Repeat Polymorphisms andIdentification of Polymorphic Short Tandem Repeats” discloses a methodincluding steps of extracting DNA from a sample to be tested, amplifyingthe extracted DNA and identifying the amplified extension products foreach different sequence. Caskey required that each different sequence bedifferentially labeled. A physical separation was performed utilizingelectrophoresis.

[0018] C. R. Cantor and others more recently disclosed a technique forscoring short tandem DNA repeats. The method is disclosed in Yarr, R. etal., “In Situ Detection of Tandem DNA Repeat Length”, Genetic Analysis:Biomolecular Engineering, 13(1996) 113-118, and PCT ApplicationW096/3673 1, PCT/US96/06527 entitled “nucleic Acid Detection Methods”.These disclose hybridization of an oligonucleotide target containingtandem repeats embedded in a unique sequence with a set of complementaryprobes containing tandem repeats of known lengths. Single-stranded loopstructures result in duplexes containing a mismatched (defined there tobe a different) number of tandem repeats. When a matched (defined thereto be identical) number of tandem repeats existed on the duplex, no loopstructure formed. The loop structures were digested with asingle-stranded nuclease. Differential wavelength, such as throughdifferentially colored fluoriflors of the various length probesidentified where matched sites existed. No express use ofelectrophoretic separation was required in accordance with this method.

[0019] Despite the knowledge of the existence of polymorphism in repeatunits now for approximately 15 years, as well as their knowndesirability for application in forensics and genetic testing,commercially acceptable implementations have yet to be achieved.

SUMMARY OF THE INVENTION

[0020] Methods and apparatus are provided for the analysis anddetermination of the nature of repeat units in a genetic target. In onemethod of this invention, the nature of the repeat units in the genetictarget is determined by the steps of providing a plurality ofhybridization complex assays arrayed on a plurality of test sites, wherethe hybridization complex assay includes at least a nucleic acid targetcontaining a simple repetitive DNA sequence, a capture probe having afirst unique flanking sequence and n repeat units, where n =0, 1, 2 . .. , being complementary to the target sequence, and a reporter probehaving a selected sequence complementary to the same target sequencestrand wherein the selected sequence of the reporter includes a secondunique flanking sequence and m repeat units, where m=0, 1, 2 . . . , butwhere the sum of repeat units in the capture probe plus reporter probeis greater than 0 (n+m>0). In accordance with this method, the sequenceof the capture probe differs at least two test sites. The hybridizationcomplex assays are then monitored to determine concordance anddiscordance among the hybridization complex assays at the test sites asdetermined at least in part by hybridization stability. Ultimately, thenature of the repeat units in the target sequence may be determinedbased upon the concordant/discordant determination coupled withknowledge of the probes located in the hybridization complex at thatsite.

[0021] By way of example, in implementation of this method, assume thata target contains six repeat units. In a system simplified merely forexpository convenience, the plurality of hybridization complex assaysmight be three assays arrayed on an APEX type bioelectronic system,wherein a first assay includes a capture probe having four repeat units(n=4), the second assay has a capture probe with five repeat units (n=5)and the third assay has capture probes with six repeat units (n=6). Ifthe reporter probe is selected to have one repeat unit (m=l), the totalnumber of repeat units at the first assay will be five (n+m=4+1=5), thetotal number of repeat units at the second assay will equal six(n+m=5=1=6), and the total number of repeat units at the third assaywill equal seven (n+m=6+1=7). The second test site will be theconcordant test site since the number of repeat units in the target inthis case equals the number of repeat units in the capture plus thereporter probes, that is it is the test site with six repeat units bothin the target and in the combination of the capture probe and thereporter probe. Utilizing the knowledge regarding probe placement, thesecond test site is known to include a capture probe having five repeatunits (n=5), such that when coupled with the knowledge of the reporterprobe including one repeat unit, the total number of six repeat units inthe target is determined.

[0022] In the preferred embodiment of these inventions, electronicallyaided hybridization or concordance and discordance determination, orboth, are utilized in the process. In one aspect, during thehybridization of the nucleic acid target with the capture probe and/orthe reporter probe, electronic stringent conditions may be utilized,preferably along with other stringency affecting conditions, to aid inthe hybridization. This technique is particularly advantageous to reduceor eliminate slippage hybridization among repeat units, and to promotemore effective hybridization. In yet another aspect, electronicstringency conditions may be varied during the hybridization complexstability determination so as to more accurately or quickly determinethe state of concordance or discordance.

[0023] In yet another aspect of this invention, a method is provided forthe determination of the nature of the repeat units in a genetic targetby providing a bioelectronic device including a set of probes arrayed ata set of test sites, the probes having a first unique flanking sequence,a second unique flanking sequence, and an intervening repeat unit serieshaving variable numbers of repeat units. The target is hybridized withthe set of probes at the set of test sites, under electronic stringencyhybridization conditions, and the concordance/discordance at the testsites is then determined. In the preferred embodiment, theconcordance/discordance is determined at least in part through the useof electronic hybridization stability determinations. The concordanttest site indicates which probe includes the number of repeat unitsidentical to that in the target. In a variation of this embodiment,electronic stringency control is utilized only during theconcordance/discordance determination.

[0024] In yet another aspect of this invention, methods and apparatusare provided for the determination of target alleles which vary in sizein a sample. A platform is provided for the identification of targetalleles which includes probes selected from the group consisting of (i)a probe having a first unique flanking sequence, an intervening repeatregion and a second unique flanking sequence, and (ii) a sandwich assaycomprising a capture probe having a first unique flanking sequence and0,1,2 . . . repeat units and a reporter probe having 0,1,2 . . . repeatunits in sequence with a second unique flanking sequence. Thereafter,the target is hybridized with the probes, preferably under electronicstringent conditions so as to aid in proper indexing, or alternatively,utilizing electronic stringency conditions during subsequent steps, orusing electronic stringency both during hybridization and at latersteps, thereafter determining concordance and discordance at the testsites as determined at least in part by hybridization stability.

[0025] In one aspect of the inventions, the location of the concordanttest site represents the nature of the target sequence repeat units bythe number of repeat units present in the target, and that in turn isbased upon the knowledge of the probes located at that test site.Namely, the particular probes associated with a given physical test sitetypically will be known in terms of their sequence, especially includingthe number of repeat units, and the physical position of those testsites results in a knowledge for the concordant sites of the nature ofthe target, especially the number of repeat units. Typically, at aconcordant test site, the number of repeat units in the target equalsthe sum of the number of repeat units in the capture probe and thenumber of repeat units in the reporter probe.

[0026] One advantageous aspect of the inventions is that the methods andapparatus are effective in determining the presence of microvariants inthe target sequence. Such microvariants may include one or moredeletions, insertions, transitions and/or transversions. These may befor a single base or for more than a single base. Deletions orinsertions within repeat units can be detected by gel separation methodswhen using highly controlled conditions. This requires single baseresolution and is near the limit of detection for most gel separationtechniques. For transitional or transversional mutations, the size ofthe allele doesn't change, even though the sequence has become altered.Conventional gel sieving methods have a very difficult time detectingthese types of mutations, and recent findings by other investigators(Sean Walsh, Dennis Roeder, DNA Forensics: Science, Evidence and FutureProspects, McLean, Va. November, 1997) suggest that transitional andtransversional mutations can cause subtle anomalies resulting indifficult gel analysis sometimes resulting in obfuscation of STRanalysis. Our method is an hybridization technique and is quite adept atreliably detecting single nucleotide polymorphisms as described above.Additionally, by designing specific capture and reporteroligonucleotides these assays can be done on the same platform used todiscriminate the nature of STR alleles by repeat unit number. Thegeneral strategy of designing capture oligos for microvariant analysisis the same as it is for integral repeat units, however reporter oligosmay differ in that they may or may not contain unique flanking sequence.The condition of effectively determining concordance by maximizing thehybridization complex stability remains since oligo design parameterswhich yield base stacking (as described above) are still followed.

[0027] In yet another aspect of these inventions, various additionalsteps may be utilized in order to promote distinguishing concordant anddiscordant test sites. One mode of concordance may be that in whichthere is a complementary match of bases in the hybridization complexincluding the capture, reporter and target in the sandwich assay format.In yet another highly advantageous arrangement, the use of juxtaposedterminal nucleotides of the reporter and capture may be utilized,wherein their contiguous nature permits interaction, such as basestacking. Advantageously, the juxtaposed terminal nucleotide identitiesmay be selected, as allowed by the existing repeat unit or otherwiserelevant sequence, so as to increase the energy difference betweenconcordance and discordance. It has been reported that base stackingbetween different bases varies in stability through an approximately4-fold range (Saenger, Principles of Nucleic Acid Structure, 1984,Springer-Verlag, New York, N.Y.). Experimental results have shown atleast a ten-fold, and often times at least more than twenty-fold,improvement in discrimination ratios for the pairings 5′G-A3′ versus5′T-A3′, when analyzed in our system. While this result is generally inconcert with the published findings that 5′G-A3′ base stacking providesgreater stability than 5′T-A3′ pairs, the differential stabilityincrease seen with our assay greatly exceeds the reported values. It ishighly beneficial that this invention exploits this natural condition toprovide a superior assay advantage. In yet other embodiments, theterminal nucleotides may be modified to increase base stacking effects,such as with the addition of propynyl groups, methyl groups orcholesterol groups. In yet another related aspect, ligation techniquesmay be utilized, such as enzyme ligation or chemical ligation, so as toincrease the energy difference between a concordant and discordant site.

[0028] Discordance may be manifested in various ways, such as in thesandwich assay format wherein a gap or overlap exists, or in the loopout method where a loop out exists. Further, discordance may exist inthe repeat region where there is a base variation, such as a deletion,insertion, transition and/or transversion.

[0029] In distinguishing concordant and discordant test sites, thedistinction is preferably drawn in part based on hybridizationstability. Hybridization stability may be influenced by numerousfactors, including thermoregulation, chemical regulation, as well aselectronic stringency control, either alone or in combination with theother listed factors. Through the use of electronic stringencyconditions, in either or both of the target hybridization step or thereporter oligonucleotide stringency step, rapid completion of theprocess may be achieved. Electronic stringency hybridization of thetarget is one distinctive aspect of this method since it is amenablewith double stranded DNA and results in rapid and precise hybridizationof the target to the capture. This is desirable to achieve properlyindexed hybridization of the target DNA to attain the maximum number ofmolecules at a test site with an accurate hybridization complex. By wayof example, with the use of electronic stringency, the initialhybridization step may be completed in ten minutes or less, morepreferably five minutes or less, and most preferably one minute or less.Overall, the analytical process may be completed in less than half anhour.

[0030] As to detection of the hybridization complex, it is preferredthat the complex is labeled. Typically, in the step of determiningconcordance and discordance, there is a detection of the amount oflabeled hybridization complex at the test site or a portion thereof. Anymode or modality of detection consistent with the purpose andfunctionality of the invention may be utilized, such as optical imaging,electronic imaging, use of charge coupled devices or other methods ofquantification. Labeling may be of the target, capture or reporter.Various labeling may be by fluorescent labeling, colormetric labeling orchemiluminescent labeling. In yet another implementation, detection maybe via energy transfer between molecules in the hybridization complex.In yet another aspect, the detection may be via fluorescenceperturbation analysis. In another aspect the detection may be viaconductivity differences between concordant and discordant sites.

[0031] In yet another aspect of these inventions, a redundant assay maybe conveniently performed. In one implementation, a serial redundantassay may be utilized, such as where after an initial hybridizationcomplex assay is performed, the stringency conditions are increased soas to effect denaturation, thereby removing the reporter from the firsthybridization complex assay. A second reporter may then be hybridized tothe remaining complex target and capture probe, wherein the secondreporter includes a number of repeat units which differs from the numberor type of repeat units in the first reporter. In this way, through thepractice of the other steps as described for other applications, thephysical test site at which concordance exists will have moved. Theresult is that a redundant assay has been performed on the same deviceand sample material.

[0032] Yet another redundant assay may be performed wherein multiple,e.g., two or more, independent sets of assays exist. A first reporter ishybridized to a first set of assays, and a second reporter is hybridizedto a second set of assays, wherein the number of repeat units in thefirst reporter differs from the number or nature of repeat units in thesecond reporter. Determination of concordance/discordance at the testsite of the arrays, when coupled with the knowledge of the probeslocated as those test sites, provides two complexes from thehybridization assays for confirmation of the target repeat number ornature.

[0033] The systems and methods of these inventions are particularlyuseful for determining the nature of complex samples, such asheterozygous samples, and mixed samples such as those from multiplesources or donors. In application, the methods and systems of theseinventions may be utilized for a broad array of applications. Among theminclude identification, such as for paternity testing or for otherforensic use. Yet another application is in disease diagnostics, such asfor the identification of the existence of a clonal tumor, where thetumor includes repeat units of a nature or number different than thepatient's undiseased genetic state.

[0034] Accordingly, it is an object of this invention to provide methodsand systems for the rapid identification of the nature and/or number ofrepeat units in a polymorphic system.

[0035] It is yet a further object of this invention to provide methodsand apparatus which may effectively provide for genetic identification.

[0036] It is yet a further object of this invention to provide systemsand methods for the accurate detection of diseased states, especiallyclonal tumor disease states, neurological disorders and predispositionto genetic disease.

[0037] It is yet a further object of this invention to provide a rapidand effective system and methods for identification, such as inforensics and paternity applications.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1A is a cross sectional view of one embodiment of an activematrix device useful in accordance with the methods of this invention.

[0039]FIG. 1B is a perspective view of an active array device usefulwith the methods of this invention.

[0040]FIG. 2 is a symbolic drawing of the components of a multiplexassay, including a target, a reporter and a capture sequence.

[0041]FIGS. 3A, 3B and 3C are diagrammatic sketches of the multiplexassay including a target, reporter and capture sequence in which thereis existing a gap, overlap and match, respectively.

[0042]FIGS. 4A, 4B and 4C show a detailed sequence listing for amultiplex assay of the TH01 locus, evidencing a gap, an overlap and amatch, respectively, corresponding to the diagrammatic representationsof FIGS. 3A, 3B and 3C.

[0043] FIGS. 5A-5G depict a diagrammatic view of a sequence of multiplexassays showing a target with eight repeat units, a reporter with asingle repeat unit and capture sequences including from four to tenrepeat units, in FIGS. 5A-5G, respectively.

[0044]FIG. 6 shows a plan view of an array of test sites for a sandwichassay, with the concordant test site depicted as shaded to represent thepresence of hybridization complex.

[0045] FIGS. 7A-7G show a diagrammatic view of a multiplexdiscrimination system, including a target having eight base repeatunits, a reporter having two base repeat units, and a series of capturesequences including from four to ten repeat units in FIGS. 7A-7G,respectively.

[0046]FIG. 8 shows a plan view of an array of test sites for a redundantassay with the concordant test depicted as shaded to represent thepresence of hybridization complex.

[0047]FIG. 9A shows a diagrammatic view of a target and hybridizedreporter in a concordant condition.

[0048]FIG. 9B shows a target and reporter in a discordant condition,namely in a loop condition.

[0049] FIGS. 10A-10G depict multiplex discrimination in a loop outsystem wherein a target includes seven repeat units and the reportersinclude from five to eleven repeat units in FIGS. 10A-10G, respectively.

[0050]FIG. 11 is a graph of fluorescence (MFI) as a function of captureoligo repeat unit number in the identification of THO1 target DNAalleles by the sandwich hybridization method.

[0051]FIG. 12 is a graph of normalized fluorescence as a function of thenumber of repeat units in the capture sequence, where the reporterincludes one repeat unit (left-hand side) and includes zero repeat units(right-hand side) showing a redundant reporter system for the THO1locus.

[0052]FIG. 13 is a chart showing the specific olignucleotides utilizedfor capture sequences, reporter sequences and the target alleles in theTHO 1 locus.

[0053]FIG. 14 is a graph of discrimination ratios as a function of G-Astacking compared to T-A stacking in the paired left, right or graphs,respectively, for an eight x versus seven x discrimination (left-handbars) and for a fifteen x versus fourteen x discrimination (right-handsbars) showing discrimination of targets by G-A and T-A stacking.

[0054]FIG. 15 is a graph of discrimination ratio showing four coupletsfor chip one through chip four, respectively, showing maximumdiscrimination ratios utilizing a ten mer reporter (a left bar incouplet) and ten mer reporter with terminal propynyl group (right bar incouplet).

[0055]FIG. 16 is a graph of normalized fluorescence intensity as afunction of capture oligo, in a heteroxygeous TPOX locus.

[0056]FIG. 17 is a table of the nucleotide sequences for the TPOXcapture oligonucleotides, reporter oligonucleotides and target allelefor the TPOX locus.

[0057]FIG. 18 is a graph of fluorescence intensity (MFI/sec) as afunction of number of capture repeat units for hybridizationdiscrimination of CSF1PON alleles.

[0058]FIG. 19 is a table of the capture oligonucleotides, reporteroligonucleotides and target alleles in the CSF1PO alleles.

[0059]FIG. 20 is a graph of normalized intensity as a function of repeatunit number in the capture sequence in a THO1/TPOX multiplex analysis.

[0060]FIG. 21 is a graph of relative fluorescence as a function ofrepeat unit number in the capture oligonucleotide in a system for theidentification of repeat unit alleles in double stranded polymers chainreaction (PCR) amplified DNA including the target THO1 locus.

[0061]FIGS. 22A, 22B and 22C are graphs of the fluorescence (MFI) as afunction of capture oligonucleotide having a gap (leftmost bar intriplet), match (center bar in triplet) or overlap (right bar intriplet) for the initial signal, for the signal after three minutes ofdenaturation and for the signal after ten minutes of denaturation forFIGS. 22A through 22C, respectively.

[0062]FIGS. 23 A and B are tables showing the discrimination of matchfrom mismatch/gap and mismatch/overlap for various combinations.

[0063]FIG. 24 is a graph of percentage fluorescence as a function ofnumber of repeat units in the capture sequence for a determination ofthe nature and number of repeat units in allelic identity of target DNAutilizing the loop out analysis.

[0064] FIGS. 25 shows a detailed sequence listing for a multiplex THO1microvariant assay.

[0065]FIG. 26 is a graph of fluorescence intensity (MFI) as a functionof capture oligonucleotide number or nature for the detection of themicrovariant allele THO19.3.

DETAILED DESCRIPTION OF THE INVENTION

[0066]FIGS. 1A and 1B illustrate a simplified version of the activeprogrammable electronic matrix hybridization system for use with thisinvention. Generally, a substrate 10 supports a matrix or array ofelectronically addressable microlocations 12. For ease of explanation,the various microlocations in FIG. 1A have been labeled 12A, 12B, 12Cand 12D. A permeation layer 14 is disposed above the individualelectrodes 12. The permeation layer permits transport of relativelysmall charged entities through it, but limits the mobility of largecharged entities, such as DNA, to keep the large charged entities fromeasily contacting the electrodes 12 directly during the duration of thetest. The permeation layer 14 reduces the electrochemical degradationwhich would occur in the DNA by direct contact with the electrodes 12,possibility due, in part, to extreme pH resulting from the electrolyticreaction. It further serves to minimize the strong, non-specificadsorption of DNA to electrodes. Attachment regions 16 are disposed uponthe permeation layer 14 and provide for specific binding sites fortarget materials. The attachment regions 16 have been labeled 16A, 16B,16C and 16D to correspond with the identification of the electrodes12A-D, respectively.

[0067] In operation, reservoir 18 comprises that space above theattachment regions 16 that contains the desired, as well as undesired,materials for detection, analysis or use. Charged entities 20, such ascharged DNA are located within the reservoir 18. In one aspect of thisinvention, the active, programmable, matrix system comprises a methodfor transporting the charged material 20 to any of the specificmicrolocations 12. When activated, a microlocation 12 generates the freefield electrophoretic transport of any charged functionalized specificbinding entity 20 towards the electrode 12. For example, if theelectrode 12A were made positive and the electrode 12D negative,electrophoretic lines of force 22 would run between the electrodes 12Aand 12D. The lines of electrophoretic force 22 cause transport ofcharged binding entities 20 that have a net negative charge toward thepositive electrode 12A. Charged materials 20 having a net positivecharge move under the electrophoretic force toward the negativelycharged electrode 12D. When the net negatively charged binding entity 20that has been functionalized contacts the attachment layer 16A as aresult of its movement under the electrophoretic force, thefunctionalized specific binding entity 20 becomes covalently attached tothe attachment layer 16A.

[0068] Before turning to a detailed discussion of the inventions of thispatent, the general matter of terminology will be discussed. The term“short tandem repeat” (STR) as used herein refers to a locus containingsimple sequence motifs which are tandemly repeated the variable numberof times at different alleles of that locus. A repeat unit or repeatunits typically refers to individual simple sequence motifs which arerepeated in a short tandem repeat. Repeat units may be, by way ofexamples, complete repeat units which contain identically repeatingsimple sequence motifs, or may be partial repeat units, such as wherethere is some difference between repeat units, such as in the existenceof microvariants between repeat units. A concordant test site is takento be a test site exhibiting a relative or local maxima of hybridizationcomplex stability. By way of example, a concordant test site may be onewherein the number of repeat units in the target is equal to the numberof repeat units in a capture plus the number of repeat units in areporter probe for a multiple system, or wherein the number of repeatunits in the target equals the number of repeat units in a probe. In yetanother example of a concordant test site, if partial repeat units arepresent, a concordant test site may be manifested by a site where therepeat units in the target are substantially similar to the nature ofthe repeat units in the capture plus probe, or single probe, asappropriate. A discordant site, on the other hand, is a site exhibitinga relatively lower level of hybridization complex stability relative toat least one other site. Examples of test sites which typically would betermed discordant would be those where there exists a gap, overlap,point mutation (e.g., single base variation such as deletion, insertion,transition and transversion), point mutations plus overlap, pointmutations plus gap, single nucleotide variants or other microvariants.

[0069] A hybridization complex assay in a multiplex system, such as in asandwich assay, typically will include a target, a capture and areporter. A hybridization complex assay in a loopout applicationincludes at least typically a target and a probe. An array as usedherein typically refers to multiple test sites, minimally two or moretest sites. The typical number of test sites will be one for each alleleof the locus. The number of loci required for a test will vary dependingon the application, with generally one for genetic disease analysis, oneto five for tumor detection six, eight, nine thirteen or more forpaternity testing and forensics. The physical positioning of the testsites relative to one another may be in any convenient configuration,where linear or in an arrangement of rows and columns.

[0070]FIG. 2 shows a symbolic drawing of the components of a multiplexassay. A target 30 includes a first unique flanking region 32, a secondunique flanking region 34 and one or more repeat units 36 disposedbetween the first unique flanking region 32 and the second uniqueflanking region 34. The target 30 may be a single or double strandednucleic acid from specific loci, such as THO1. A reporter 40 includes atleast a unique sequence region 42, and optionally includes one or morerepeat units 44 disposed at the terminal end of the unique sequenceregion 42. The reporter 40 may have no repeat units 44, or may includeone or more repeat units 44. If the reporter 40 is to be labeled, alabel 46 is associated therewith. Typically, the unique sequence region42 of the reporter 40 is complementary to the second unique flankingregion 34 of the target 30. The capture 50 includes a capture uniquesequence region 52 and one or more repeat units 54 which are adjoined tothe terminal end of the capture unique sequence region 52. If thecapture 50 is to be attached to a solid support or other anchoringmedium, an attachment element 56, such as biotin may be utilized.

[0071]FIGS. 3A, 3B and 3C are diagrammatic sketches of the multiplexassay including a target, reporter and capture sequence in which thereexists a gap, overlap and match, respectively. For simplicity, thenumbering of FIG. 2 will be adopted here for corresponding structures.In FIG. 3A, a gap condition exists. Broadly, the target 30 is hybridizedto the reporter 40 and to the capture 50. More particularly, the secondunique flanking region 34 is hybridized to the complementary strandcomprising the unique sequence region 42 of the reporter 40. Similarly,the first unique flanking region 32 of the target 30 is hybridized tothe complementary capture unique sequence region 52 of the capture 50.The target 30 includes eight repeat units 36 in this example. Thestructure as best described applies equally to FIGS. 3A, 3B and 3C. FIG.3A depicts a gap region 56, which results from the capture 50 having sixrepeat units 54 and the reporter 40 having one repeat unit 44. Thus, thetotal number of repeat units 44, 54 in the reporter plus capture isseven, which is one less than the total number of repeat units 36 in thetarget 30. In FIG. 3B, an overlap condition is shown. Here, the captureincludes eight repeat units 34, and the reporter 40 still includes asingle repeat unit 44. Here, the total number of repeat units 34, 44between the capture 50 and reporter 40 is nine, exceeding the number ofrepeat units 36 in the target 30, whereby one repeat unit is overlapped,here shown to be the repeat unit 44 associated with the reporter 40.FIG. 3C shows a match between the target 30 and the reporter 40 pluscapture 50. There are seven repeat units 34 associated with the capture50 and one repeat unit 44 associated with the reporter 40. Accordingly,the number of repeat units 36 in the target 30 equals the sum of therepeat units 34 in the capture 50 plus the number of repeat units 44 inthe reporter 40.

[0072]FIGS. 4A, 4B and 4C show one example of specific nucleotidesequences corresponding to the examples of FIGS. 3A, 3B and 3C. Notethat the left to right orientation of FIGS. 3A, 3B and 3C is reversedleft to right for FIGS. 4A, 4B and 4C. FIG. 4A shows a gap conditionwherein the gap 56 is disposed between the repeat unit 44 of thereporter 40 and the terminal repeat unit (5′ CATT3′) adjacent the gap 56of the capture 50. In FIGS. 4A, 4B and 4C, the base repeat unit 36 ofthe target 30 is 5′AATG3′, and accordingly, its complement base sequenceis 5° CATT3′ 44, 54. In FIGS. 4A, 4B and 4C, the nucleotides of the baserepeat unit are shown in capital letters. This is done to designatethose units in distinction to the nucleotides forming the first uniqueflanking region 32 and second unique flanking region 34 of the target 30as well as the unique region sequence 42 of the reporter 40 and thecapture unique sequence region 52 of the capture 50. As can be seen, thenucleotides in the hybridized strands are paired, namely A-T and G-Cpairs are complementary. FIG. 4B shows a mismatch with an overlap of onerepeat unit. Specifically, the base repeat unit 44 comprising the series5′ CATT3′ is displaced from a hybridized condition with the base unit 36adjacent the second unique flanking region 34 of the target 30. Asshown, the 5′ terminal nucleotide (designated “c”) of the uniquesequence region 42 of the reporter 40 is shown as being slightlydisplaced from complete hybridization with the complementary “g”terminal nucleotide of the second unique flanking region 34 of thetarget 30. This depiction is optional, and may also include thecondition in which the terminal nucleotide of the unique sequence region42 is in a hybridized condition with the terminal nucleotide of thesecond unique flanking region 34 of the target 30. FIG. 4C shows a matchcondition, where in this example, the nature of the repeat region, thatis both number of repeat units partial and whole, and thecomplementarity of the sequence match., In the matched condition of FIG.4C, the 5′ terminal nucleotide “C” of the repeat unit 44 of the reporter40 is adjacent and contiguous with the 3′ terminal nucleotide “T” of thebase repeat unit 54 of the capture 50, permitting base stacking betweenthe 5′ “C” of the repeat unit 44 of the reporter 40 and the 3′ “T” ofthe base repeat unit 54 of the capture oligonucleotide 50. The two basestacked nucleotides are underlined in FIG. 4C. In a preferredembodiment, the hybridization complex may anchored via the capture oligo50 which would contain the appropriate attachment chemistry, preferablybiotin at its 5′ terminus. Also in a preferred embodiment, thehybridization complex would be labeled via the reporter oligo 40 with anappropriate molecule, preferably a chromophore at it's 3′ end.

[0073] The nature of a repeat unit is defined here as comprised of boththe number of whole (or integral) repeat units and partial repeat units.Partial repeat units also known as microvariants or cryptically simplesequence, may be comprised of single nucleotide divergences from themost common repeat unit sequence. These divergences may consist ofinsertions, deletions, transition or transversion polymorphisms of thesimple repeat sequence. Since all prior methods for the analysis of STRloci have relied on size, or the number of nucleotides, information onthe frequency of transition and transition repeat unit polymorphisms isscant. However other investigators have recently recognized theirsignificance and it is likely that methods which can efficiently detectthem will be valuable (Sean Walsh and Dennis Roeder, DNA Forensics,Science, Evidence and Future Prospects, McLean, Va., November 1997).

[0074]FIG. 25 demonstrates how this invention detects the presence of acommon microvariant TH01 9.3. Two new entities are presented here: amicrovariant target sequence 70 containing the partial repeat unit 71ATG and a microvariant reporter oligonucleotide 80 which iscomplementary to 71 and the seven 5′ adjacent nucleotides. FIG. 25 showsthe relationship of the DNA subsequences when the nature of the targetallele is nine whole repeat units and one partial repeat, resulting in amatched concordancy. Elements which have been discussed before in FIG.4A-4C have retained their numerical appellation, and novel features havebeen labeled with new numbers. Therefore the target sequence 70 is madeup of a first unique sequence 34, integral repeat sequence 36, secondunique flanking sequence 32 and presents partial repeat sequence 71. Thecapture sequence 50 is identical to that described in FIG. 4A, with theexception that it has only three repeat units 54. The microvariantreporter 80 is similar to reporter 40 in that it has repeat sequence 44but differs by a lack of unique flanking sequence and by the inclusionof sequence 81 which is complementary to the target 70 partial repeatunit 71. The reporter 80 stability is enhanced by two features. First itis complementary only to the microvariant region and second, it willbase stack and therefore attain concordancy or a local maxima ofstability only at the site which contains the 3ru capture oligo. Onepracticed in the art will realize how to apply this invention tomicrovariant sequences which differ from the TH01 9.3 sequence. FIG. 26demonstrates the effectiveness of this method.

[0075] Selection of the adjacent or proximal nucleotides so as toincrease the energy difference between concordant and discordant testsites is advantageously employed. A detailed discussion of suchselections or modifications, such as in the use of terminal nucleotidebase stocking, or modifications of terminal nucleotides such as withpropynyl groups, methyl groups or cholesterol groups, or through the useof ligation techniques such as enzymatic ligation or chemical ligationare discussed further, below.

[0076] FIGS. 5A-5G depict a diagrammatic representation of a multiplexsystem. The hybridization complex used in this system is sometimestermed a sandwich assay. Again, for expository convenience, thenumbering adopted corresponds to that utilized with FIG. 2, FIGS. 3A, 3Band 3C and FIGS. 4A, 4B and 4C. In each of the depictions, a target 30has a unique flanking region 32 and a second unique flanking region 34,with an intervening set of repeat units 36. In the example, the numberof repeat units 36 is 8. The reporter 40 includes a unique sequenceregion 42 which is complementary to the second unique flanking region34, and includes in this example one repeat unit 44 at the terminal endof the reporter. The capture 50 includes a capture unique sequenceregion 52 and, in this example, multiple repeat units 54 at the terminalend of the capture 50. The capture unique sequence region 52 iscomplementary to the first unique flanking region 32.

[0077]FIG. 5A shows a capture 50 with 4 repeat units 54. Since the sumof the number of repeat units 54 in the capture 50 plus the number ofrepeat units 44 in the reporter 40 (4+1) is less than the number ofrepeat units 36 in the target 30 (eight) a gap 56 exists. The gap asshown in FIG. 5A is substantially of the length of 3 repeat units 36,44, 54. As a matter of terminology, the number of repeat units 54 in thecapture 50 is sometimes denominated an “N capture”, where N equals thenumber of base repeat units 54 in the capture 50 plus the number of baserepeat units 44 in the reporter 40. With this terminology, a matchexists with a N capture where N equals the number of repeat units 36 inthe target 30. Thus, in the example of FIG. 5D, wherein a matchcondition exists, a capture 50 having 7 repeat units 54 may be alsodenominated an “8 capture” since the capture 50 having 7 repeat units 54when used with these selected reporter 40 having a single repeat unit 44provides a match in that the total number of repeat units 44, 54 equalsthe number of repeat units 36 in the target. It will be appreciated bythose skilled in the art that various naming or number conventions maybe utilized to accurately describe the underlying arrangements, and itis those underlying arrangements which comprise the inventions herein,and not the naming or numbering conventions adopted.

[0078]FIG. 5B shows a capture 50 having 5 repeat units 54, wherein a gap56 of the length of substantially 2 repeat units 36, 44, 54 exists. FIG.5C shows a capture 50 having 6 repeat units 54, wherein a gap 56 ofsubstantially the length of a single repeat unit 36, 44, 56 exists. FIG.5D shows a match condition with a capture 50 having 7 repeat units 54and a reporter 40 having a single repeat unit 44 equals the number ofrepeat units 36 in the target 30.

[0079]FIGS. 5E, 5F and 5G show an overlap condition. FIG. 5E shows acapture 50 with 8 repeat units 54. As depicted, the reporter 40 repeatunit 44 is shown as being in a substantially non-hybridized conditionwith the target 30. FIG. 5F shows a capture 50 with 9 repeat units 54,wherein a terminal repeat unit 58 of the capture 50 and the repeat unit44 of the reporter 40 are both in a substantially non-hybridizedcondition with respect to the target 30. FIG. 5G shows a capture 50having 10 repeat units 54, such that the two terminal repeat units 58 ofthe capture 50 and the repeat unit 44 of the reporter 40 are in asubstantially non-hybridized relationship with the target 30.

[0080]FIG. 6 shows a plan view of an array of test sites for use in amultiplex assay, such as a sandwich assay. The concordant test site isdetermined to be at the site containing the 7 repeat unit capture. Thisfigure depicts an assay done with a 1 repeat unit reporter, thereforeone can determine that the target must contain 8 repeat units since atthe concordant site, the number of repeat units in the capture (7) plusthe number of repeat units in the reporter (1) equals 8. The depictionrelates to the diagram of FIG. 5 in that it shows the results attainedin the analysis of a DNA sample containing an eight repeat unit targetwith a one repeat unit reporter.

[0081]FIGS. 7A through 7G are diagrammatic depictions of a multiplexsystem, such as a sandwich assay, in which the reporter includes tworepeat units. This is in distinction to the assay of FIGS. 5A-G whereinthe reporter included a single repeat unit. Again, for expositoryconvenience, the numbering of earlier figures will be adopted to theextent of similarity. FIG. 7A shows a target 30 having a first uniqueflanking region 32 and a second unique flanking region 34. The reporter40 includes a unique sequence region 42 and, in this example, two repeatunits 44. The capture sequence 50 includes a capture unique sequenceregion 52 and 4 repeat units 54. Again, as a matter of nomenclature, thecapture 50 may be referred to as a “5 capture”, reflecting theterminology utilized in connection with the assay of FIGS. 5A-5G, namelythose in which a reporter 40 having a single repeat unit 44 is utilized.

[0082]FIG. 7B shows a multiplex system wherein the capture 50 includes 5base units 54. Each of the examples of FIG. 7A and 7B include a gap 56.

[0083]FIG. 7C shows a match condition in that the number of repeat units36 in the target 30 is equal to the sum of the number of repeat units 54in the capture 50 plus the repeat units 44 in the reporter 40 (6+2=8).FIG. 7C depicts the concordant test site in that the match conditionexists. Note that the effective location of the concordant test site hasshifted from FIG. 5D to FIG. 7C. This is a reflection of the change inthe number of repeat units 44 in the reporter 40. Where a sequence orflight of unit incrementally longer captures 50 are utilized, a reporter40 being one base unit 44 shorter or longer will shift the physicallocation of the concordant test site, such as in FIG. 6 from test site26D to 26C when going from a reporter of one repeat unit 44 to areporter 40 having two repeat units 44.

[0084] FIGS. 7B-7G show an overlap condition. In FIG. 7D, one repeatunit 44 of the reporter 40 is in a hybridized relationship with a repeatunit 36 of the target 30. A second repeat unit 44′ of the reporter 40 isin a mismatched, overlap condition with the complex. FIG. 7E shows anoverlap condition wherein repeat units 60 are in a non-hybridizedcondition with the target 30. FIG. 7F and 7G also include mismatcharrangements including overlap wherein multiple repeat units 60 are in anon-hybridized condition with the target 30. The mismatch repeat unitsmay either be those from the reporter 30 or from the capture 50, or acombination of both.

[0085]FIG. 8 shows a plan view of an array of test sites for use in amultiplex assay, such as a sandwich assay. The concordant test site isdetermined to be at the site containing the 6 repeat unit capture. Thisassay depicts an assay done with a 2 repeat unit reporter, therefore onecan determine that the target must contain 8 repeat units since at theconcordant site, the number of repeat units in the capture (6) plus thenumber of repeat units in the reporter (2) equals 8. The depictionrelates to the diagram of FIG. 7A-7G in that it shows the resultsattained in the analysis of a DNA sample containing an eight repeat unittarget with a two repeat unit reporter. In comparison with FIG. 6, onenotes the change in the concordant test site location and confirmationof the target allele determination, when the target DNA is redundantlyassayed with a second reporter oligonucleotide.

[0086]FIG. 9A and FIG. 9B show diagrammatic views of a loopoutembodiment of the invention. In the figures, a capture 60 includes afirst unique flanking sequence 62, a second unique flanking sequence 64and an intervening sequence of repeat units 66 comprising one or morerepeat units. A reporter 70 includes a reporter first unique flankingsequence 72, a reporter second unique flanking sequence 74 and anintervening sequence of repeat units 76. The number of repeat units inthe intervening sequence of repeat units 76 of the reporter 70 may bethe same as or different than the number of repeat units in theintervening sequence of repeat units 66 of the capture 60. A reporterlabel 78 may be included.

[0087]FIGS. 10A through 10G are depictions of multiplex systems having avariable length of repeat units. The numbering for FIGS. 7-10 will adoptthat from FIGS. 9A and 9B to the extent practicable. In FIG. 10A, acapture 60 includes a first unique flanking sequence 62, a second uniqueflanking sequence 64 and an intervening sequence of repeat units 66having 5 repeat units. The reporter 70 includes a reporter first uniqueflanking sequence 72, a reporter second unique flanking sequence 74 anda intervening sequence of repeat units, specifically, 7 repeat units. Amismatch or loopout condition exists given the different number ofrepeat units in the intervening sequences 66, 76. Similarly, in FIGS.10B and 10D-10G, a mismatch or loopout condition exists. Each of thecomponent figures includes 7 base repeat units in the reporter 70. Aflight or sequence of monitonically increasing number of repeat units inthe intervening sequence of repeat units 66 for the capture 60 isdepicted. FIG. 10B includes 6 repeat units, which still results with amismatch, loopout condition in FIG. 10B where the excess repeat units inthe intervening sequence 76 of the reporter 70 are looped out. In eachof FIGS. 10D-10G, the number or repeat units in the intervening sequence66 in the capture 60 exceeds the number of repeat units in theintervening sequence 76 of the reporter. A loopout or mismatch conditionthen exists. In FIG. 10D, 8 repeat units exist within the interveningsequence of repeat units 64, which differs by one repeat unit from thenumber within the reporter intervening sequence of repeat units 76. InFIG. 10E, there are 9 repeat units in the intervening sequence of repeatunits 64 in the capture 60. In FIG. 10F, there are 10 repeat units inthe intervening sequence of repeat units 64 of the capture 60. In FIG.10G there are 11 repeat units in the intervening sequence of repeatunits 64 of the capture 60.

[0088] In one mode, the hybridization complex is labeled and the step ofdetermining concordance and discordance includes detecting of theamounts of labeled hybridization complex at the test sites. Thedetection device and method may include, but is not limited to: opticalimaging, electronic imaging, imaging with a CCD camera and integratedoptical imaging. Further, the detection, either labeled or unlabeled, isquantified, which may include statistical analysis. The labeled portionof the complex may be the: target, capture, reporter or thehybridization complex in toto. Labeling may be by fluorescent labelingselected from the group of but not limited to: Bodipy Texas Red, BodipyFar Red, Lucifer Yellow, Bodipy 630/650-X, Bodipy R6G-X and 5-CR 6G .Labeling may further be done by colormetric labeling, bioluminescentlabeling and/or chemiluminescent labeling. Labeling further may includeenergy transfer between molecules in the hybridization complex by:perturbation analysis, quenching, electron transport between donor andacceptor molecules, the latter of which may be facilited by doublestranded match hybridization complexes (See, e.g., Tom Meade and FaizKayyem, electron transport through DNA). Optionally, if thehybridization complex is unlabeled, detection may be accomplished bymeasurement of conductance differential between double stranded and nondouble stranded DNA (See, e.g., Tom Meade and Faiz Kayyem, electrontransport through DNA). Further, direct detection may be achieved byporous silicon-based optical interferometry.

[0089] The label may be amplified, and may include for example branchedor dendritic DNA. If the target DNA is purified, it may be unamplifiedor amplified. Further, if the purified target is amplified and theamplification is an exponential method, it my be, for example, PCRamplified DNA or SDA amplified DNA. Linear methods of DNA amplificationsuch as rolling circle or transcriptional runoff may be used. Whereintarget DNA is unpurified and unamplified or amplified, the amplificationmethods further consisting of PCR and SDA for exponential amplificationand rolling circle or transcriptional runoff for linear amplification.

[0090] The target DNA may be from a source of tissue including but notlimited to: hair, blood, skin, sputum fecal matter, semen, epithelialcells, endothelial cells, lymphocytes, red blood cells, crime sceneevidence. The source of target DNA may include: normal tissue, diseasedtissue, tumor tissue, plant material, animal material, mammals, humans,birds, fish, microbial material, xenobiotic material, viral material,bacterial material, and protozoan material.

[0091] Wherein the target material is from cloned organisms (Ian Wilmut,Roslyn Institute, Edinborough) to determine degree of identity and levelof genetic drift.

[0092] Further, the source of the target material may include RNA.Further yet, the source of the target material may include mitochondrialDNA.

EXAMPLES

[0093] Example 1

Identification of TH01 Target DNA Alleles by the Sandwich HybridizationMethod

[0094] The TH01 locus contains the tetranucleotide repeat (AATG) presentin five to eleven copy-numbers in a noncoding region of the HumanTyrosine Hydroxylase gene (ref). This locus is one of many commonly usedand accepted by the forensics community for DNA fingerprinting. FIG. 1depicts data from an experiment designed to determine the identities ofthe alleles present in an unknown target DNA sample after analysis bythe method described here.

[0095] A silicon chip was prepared by spin coating onto the top of theelectrodes an organic layer of agarose mixed with streptavidin, thusforming the permeation layer that serves as the underlying foundationfor DNA attachment (See e.g., U.S. application Ser. No. 08/271,882,filed Jul. 7, 1994, entitled “Methods for Electronic Stringency Controlfor Molecular Biological Analysis and Diagnostics”, accord., Sosnowskiet al., 1997, Proceedings of the National Academy of Science USA). Thispermeation layer is contiguous with the electrode on one side andcontiguous with the buffer containing the analyte on the other. CaptureDNA specific for each TH01 allele was then electronically addressed toindividual sites on the spin coated chip, so that each test site iscapable of detecting a different TH01 allele. Sequences for the captureoligos are listed in Table 1. The capture oligos were electronicallyaddressed in 50 mM histidine buffer at its natural pH ˜5.4, at aconcentration of 500 nM. Pads were biased positive 5 at a time and acurrent source of +4.0 microamps (μA) was applied for 38 milliseconds(ms). The polarity of the field was then reversed and −4.0 ˜ A wasapplied to the 5 pads for 25 ms. This cycle was repeated 500 times for atotal electronic addressing time of˜30 seconds. Under these conditions,the biotin moiety of the capture oligo reacts with the streptavidin inthe permeation layer over the activated test site to immobilize thecapture oligo at that site.

[0096] A mix of complementary target DNA, composed of TH01 alleles 5 and9 (Table 1), was then electronically hybridized to each of the sitescontaining addressed capture DNA. The electronic hybridization was donein low conductivity zwitterionic buffer at a temperature empiricallydetermined to promote nonslippage hybridization. Due to the nature ofelectronic hybridization, specifically the low conductivity buffer(Edman, et al., Nucleic Acids Research, 1997), high stringencyhybridization can be attained at lower temperatures than conventionalnonelectronic hybridization. Experiments for TH01 analysis were usuallyperformed at 34-42% C. The target DNA was electronically hybridized in50 mM histidine buffer at its natural pH˜5.4, at a concentration of5-125 nM. The programmed electronic protocol included the followingsteps. Pads were biased positive 5 at a time and a current source of+4.0 microamps (μA) was applied for 19 milliseconds (ms). The polarityof the field was then reversed and −4.0 μA was applied to the same 5pads for 12 ms. This biased-AC cycle was repeated 500 times for a totalelectronic addressing time of 30 seconds.

[0097] This experiment has also been done by passive, nonelectronichybridization at high stringency conditions, but with much longerincubation times (50 mM NaPO₄ buffer, pH 7.0, 60% C, 30-60 minutes,results not shown).

[0098] The stringency of this hybridization step is critical due to themalleable nature of the tetranucleotide repeat complementary alignment.It is quite easy to obtain stable hybrids without aligning the flankingunique sequences, since the length of the repeat region is 20-44 bases.The out of register hybrids formed by insufficient stringency will notbe accurately distinguished by any hybridization assay. High stringencyhybridization can be attained at relatively low temperatures withelectronic hybridization because of the low conductivity of the bufferand resulting low shielding of the repulsive negative charges on the DNAbackbone. Electronic concentration of DNA overcomes these repulsiveeffects while maintaining highly stringent hybridization conditions.

[0099] Reporter DNA, 1 Repeat Unit (FIG. 13, 500 nM in 50 mM NaPO4, 500mM NaCl, pH 7.0) was then passively hybridized to the capture-targetcomplexes formed by the above steps. Reporter hybridization was moststable at those test sites where the target directs hybridization toprovide a juxtaposition of the terminal nucleotides of the capture andreporter oligo. This additional stability is due to the base stacking ofthe terminal nucleotides. This juxtaposition will be 5′-3′ or 3′-5′depending on the position of the attachment chemistry on the captureoligo. Unstable configurations would be a four base (or greater) gapbetween the capture and reporter or a four base (or greater) overlap ofthe capture and reporter ( See, e.g., FIGS. 3A-3C, and 5A-5G).

[0100] After reporter hybridization, the DNA loaded chip is washedseveral times with 50 mM NaPO_(4, pH) 7.0 at ambient temperature. Thetemperature of the hybridized organo-chip is then increased to 30% C andfluorescence levels at each test site are recorded at one minute timeintervals. The fluorescent values are digitized by a computer program(IP Lab) as mean pixel intensity. A specific area over each pad isselected, and the pixel intensity for each site is stored for analysis.The histogram in FIG. 1 displays the mean pixel intensity at each testsite immediately after the denaturation step is complete.

[0101]FIG. 11 shows a graph of the fluorescence as a function of numberof repeat units. These results show that a heterozygous mix of TH01 DNAcan be resolved into match (concordant) and mismatch (discordant)hybrids, with the match hybrids representing the identity of the allelespresent in the DNA sample. All possible homozygote and heterozygote TH01STR allelic combinations (5+6, 5+7, 5+8 etc.) have been analyzed by thechip format, such as shown in FIGS. 1A and 1B, with similar excellentlevels of discrimination among alleles.

Example 2 Reanalysis of Target DNA with Redundant Reporters

[0102] One Repeat Unit reporter was denatured from the match sites ofthe chip described in the preceding example by increasing thetemperature ˜50% C, conditions which do not denature the target from thecapture. This chip was then rehybridized with the Zero Repeat Unitreporter ( See, e.g., FIG. 5). This shifts the position of stablesandwich complexes from the 4 and 5 sites (See, FIG. 12, left hand side,One Repeat Unit Reporter) to the 5 and 6 sites (FIG. 12, right handside, Zero Repeat Unit Reporter). Using the formula that the number ofrepeat units in the capture plus the number of repeat units in thereporter equals the number of repeat units in the target, we find thatthe target DNA in this case had a heterozygous mix of the 5 and 6alleles of TH01. The reanalysis confirms the identity of the allelespresent in the target DNA with a second oligo sequence. This redundantanalysis increases the significance of the assay result since it isessentially a new interrogation of the target DNA with an oligo that hasa different sequence. Using a different sequence reduces the possibilityof artifactual results due to oligo secondary structure or othersequence-related anomalies. Therefore, the use of additional oligos fortarget analysis reduces the possibility of false positive and/ornegative results.

[0103] The above protocol was repeated with the Two Repeat Unit reporter( See, e.g., FIGS. 7A-7G), to shift the location of stable match hybridsto a third test site. This additional reiteration of the STR analysisfurther strengthens the robustness of the assay.

Example 3 Selection and/or Modifications of Terminal Nucleotides to

[0104] Increase Base Stacking Effect

[0105] Base stacking is dependent on the interactions of the ringstructure of one base with the base ring of its nearest neighbor. Thestrength of this interaction depends on the type of rings involved, asdetermined empirically. While applicants do not wish to be bound by anytheory, among the possible theoretical explanations for this phenemonare the number electrons available between the two bases to participatein pi bond interactions and the efficiency of different basecombinations to exclude water from the interior of the helix, therebyincreasing entropy, Although the above models are consistent withcurrent data, the possible mechanisms of stacking interactions are notlimited to these concepts.

[0106] It has also been observed that modification of bases involved inbase stacking interactions can strengthen pi bonding, or stacking,between them. As one might predict from the models described above,these modifications provide more electrons for use in Pi bonding and/orto increase the surface area of the rings thereby increasing the area ofhydrophobicity between the stacked bases.

[0107]FIG. 14 demonstrates an example of these models as applied to thisinvention. Initial experiments with the CSF1PO locus used an A and T asthe terminal nucleotides to provide discriminating base stacking.References indicate that A-T base stacking interactions are the leaststable of all nucleotide combinations. Therefore we altered the designof the capture and reporter oligos to make G and A the terminalnucleotides, since this is reported to be a much more stableconformation. The experiment was done by the method described in Example1, with the exception that the locus examined was CSF1PO. To compare thebase stacking contributions of different juxtaposed contiguous terminalnucleotides, an additional set of CSF1PO capture and reporter weredesigned to change the terminal nucleotides from T-A to G-A. FIG. 15compares discrimination of match from mismatch hybrids containing eitherA-T or G-A terminal nucleotides. The results are displayed asdiscrimination ratios, that is the Mean Fluorescent Intensity (MFI) ofthe concordant site divided by the average MFI of the discordant sites.One sees that the Discrimination Ratios increase from about 2.5 to about25 when G-A terminal nucleotides are used rather than T-A terminalnucleotides. These data demonstrate that this system can be modulated ina manner predicted by base stacking theory, as well as earlierobservations, thereby underscoring the mechanism of the invention asdependent on Pi bonding between juxtaposed bases.

[0108] In addition to taking advantage of the naturally selected basestacking interactions, it may be predicted that base modifications whichincrease the number of electrons in the ring or enlarge the hybdrophobicarea would also increase discrimination of match from mismatch hybrids.This was tested by synthesizing TH01 reporter oligos whose 5′ terminalnucleotide contained a propynyl group attached to the ring of the base.This modification would be predicted to increase base stacking by eitherof the increased electronorhydrophobicity models described above. FIG.15 shows match/mismatch results in a direct comparison of a TH01reporter with or without a propynyl-modified terminal base. Thisexperiment was done as described in Example 1 with the TH01 locus. Thedata are again presented as discrimination ratios. In 4 separateexperiments, enhanced stability is observed in complexes containingreporters with propynyl-modified reporters. The average increase indiscrimination ratios was 95%. The results show that again, this systemcan be manipulated in a predictable fashion. This concept could becarried further by adding other analogs such as methyl of cholesterolgroups. Techniques for adding these types of modifications are known(e.g. Gryaznov).

[0109] These modifications could be used to further stabilize thebinding of the reporter to the concordant by linking the modifyingmolecules together. One example of this is taught by Gryaznov in the useof cholesterol at both terminal nuclei and the addition of a cholesterolbinding molecule, such as low density lipoprotein (LDL). This wouldresult in a complex at the concordant site which consists of target,cholesterol-modified capture, cholesterol-modified reporter and LDL.

Example 4 Hybridization Detection of TPOX alleles

[0110] The TPOX locus contains the tetranucleotide repeat (AATG) presentin six to thirteen copy-numbers in a noncoding region of the HumanThyroid Peroxidase gene (See, e.g., Anbach et al., 1996, Advanced inForensic Haemogenetics). This locus is also one of many commonly usedand accepted by the forensics community for DNA fingerprinting. Thesequences are provided in FIG. 17.

[0111]FIG. 16 depicts data from an experiment where target DNAcontaining the TPOX 8 and 11 alleles was analyzed by a procedure nearlyidentical to that described in Example 1. Oligo capture DNA containingall allelic possibilities was electronically addressed to individualsites on the chip. A mix of complementary target DNA, composed of TPOXalleles with 8 and 11 STRs, was then electronically hybridized to eachof the pads containing addressed capture DNA. The conditions forelectronic hybridization were the same as those outlined in Example 1.One Repeat Unit Reporter oligo was then passively hybridized to thearray and treated in the manner of the TH01 example. FIG. 16 shows astable hybridization complex at the test sites containing capture oligoswith 7 and 10 repeat units. Since the reporter oligo has one repeatunit, the target DNA can be identified as having 8 and 11 repeat units.

[0112] The results show that a mix of TPOX 8 and 11 STR DNA can beunequivocally discriminated from all mismatches. Further, all otherhomozygous and heterologous TPOX combinations analyzed yield comparablediscrimination.

Example 5 Hybridization discrimination of CSF1PO alleles

[0113] Capture oligos containing CSF1PO alleles 7 through 15 (FIG. 19)inclusive were electronically addressed to representative sites aspreviously described. Target DNA containing CSF1PO 11 and 12 alleles(FIG. 19) was then electronically hybridized to each of the sites. TheCSF1PO one repeat unit reporter ( FIG. 19) was then. Denaturation of thereporter was done at 30% C. FIG. 18 shows the mean pixel intensity atvarious capture sites after the assay, demonstrating the ability of theassay to correctly discriminate the alleles present in the targetsample. The experiment was done as described in Example 1.

Example 6 TH01/TPOX Multiplex Analysis

[0114] Locus-allele specific capture oligos were individually addressedto different sites on a single chip. The DNA chip containing captureoligos was then hybridized with a mixture of TH01 and TPOX target DNAcontaining heterozygote alleles. The chip was then washed and analyzedby the hybridization assay of the form described previously. The abovesteps were performed as described in Example 1. Relative fluorescentlevels were used to determine whether sites contain concordant ordiscordant DNA hybrid complexes. Both reporters used contained onerepeat unit.

[0115] The results of FIG. 20 showed that under the assay conditions, 7and 9 STR alleles of THO1 hybridized very well with their cognitivecapture sites. Hybridization to other capture alleles was not detectable(5 x, 7 x, 9 x and 10 x), indicating an excellent discrimination of TH017/9 heterozygote. For the TPOX locus, we also obtained a good matchedcapture/target interaction (sites 9 x and 11 x). Further, the stabilityof the discordant hybridization complexes formed with the 10 and 12 STRtargets was so low that the complexes were either undetectable (7 xc and12 xc), or low enough to yield a discrimination ratio of 15 fold orhigher (10 xc and 8 xc respectively), resulting in easy discriminationof TPOX 10/12 heterozygote target.

Example 7 Identification of STR Alleles in Double Stranded PCR-amplifiedDNA

[0116] This experiment was done to determine the utility of the currentinvention as applied to interrogation of double-stranded DNA generatedby PCR amplification. FIG. 6 provides an example of the ability of oursystem to accurately identify PCR generated targets.

[0117] The TPOX 1 locus was PCR amplified using a genomic template fromthe K562 cell line following standard conditions outlined in the PromegaSTR User's manual (3). The genotype of K562 is heterozygous for the 8and 9 repeat alleles. Following amplification the amplicon was denaturedat 95% C and hybridized to a Nanogen APEX chip. As previously discussed,the chip had capture probes unique for PCR products containing eachnumber of repeat length.

[0118] The technical aspects of this experiment were identical to thosedescribed in Example 1 and 4, with the exception of the use ofdouble-stranded, PCR amplified DNA as the target.

[0119]FIG. 21 shows the relative amount of signal present on thepositive (8C, 9C match) and negative (7C, 10C mismatch) after theexperiment has been performed. As seen in example 4 the level ofdiscrimination attainable ranges from 20-fold to infinite. Similarresults have been obtained using CSF1 and TH01 from both K562 controlDNA and genomic DNA isolated from anonymous donors. These resultssuggest that the current invention is generally applicable to alldouble-stranded DNA, whether amplified by PCR or other technology,including the potential for analysis of unamplified DNA.

Example 8 Multivariant Detection

[0120] In the examples listed previously, detection has beenaccomplished by direct fluorescent labeling of the reporter orreporter/target DNA. One embodiment would be fluorescence perturbationwhere quencher and reporter chromophores are positioned proximal to eachother such that fluorescence is quenched. See, e.g., (Methods forHybridization Analysis Utilizing Electrically Controlled Hybridizationand Methods For Electronic Fluorescent Perturbation for Analysis andElectronic Perturbation Catalysis for Synthesis), all incorporated byreference as if fully set forth herein.

[0121] Oligo synthesis and conjugation methods and materials arecommonly practiced. In brief, the capture probe would have attachmentmoiety, such as biotin, at one end and a chromophore at the distalterminus or the terminus which extends into the STR region. During DNAsynthesis linker arms or spacers would be incorporated at theappropriate location internally or at the terminus. These linker armswould have a functional group to which chromophores could later beconjugated, such as amino linker and succinimidyl ester chromophore. Thereporter probe would have a different chromophore incorporated in thesame manner, at the end which extends into the STR region. Thus, in thepresence of target the capture and reporter probes would hybridize andposition the chromophores proximal to one another. The distance betweenthe chromophores would be determined by the spacer length and where thechromophore was attached to the DNA, via the base, backbone, or sugar.

Example 9 Target -Dependent Ligation of Capture and Reporter

[0122] An additional embodiment of this invention would be to furtherstabilize the attachment of the reporter to the concordant test site byligating the reporter to the preattached capture. This would result in acovalent bond between the capture and the reporter with the capturebeing held at the site by biotin-streptavidin interaction.

[0123] The critical part of this embodiment would be to accomplish theligation in a selective manner, maintaining the ability to discriminatematch from mismatch hybrids. This could be done by careful maintenanceof hybridization stringency, by electronic or conventional methods.

[0124] Ligation could be achieved enzymatic (Maniatis et al., MolecularCloning, a Laboratory Manual, 1982) or chemical methods (Gryaznov,Nucleic Acids Research, 1994). Selection of the method could bedetermined by the kinetics involved with the specific type of reactionas well as the overall efficiency of taking a particular method into aproduct.

Example 10 Discriminating Match from Mismatch/Gap and Mismatch/Overlap

[0125] The ability of the system to discriminate not only match frommismatch hybrids, but also to discern between the two types ofmismatches, gap and overlap increases the utility of the method. FIGS.22A, 22B and 22C show graphics of fluorescence intensity for a gap,match and overlap condition (bar charts from left to right), for theinitial signal (FIG. 22A) after three minutes of denaturation (FIG. 22B)and after ten minutes of denaturation (FIG. 22C). This feature of thetechnology provides additional information about the target DNA, thatis, information regarding all three types of possible hybrids. Thisadditional information can be used in several ways.

[0126] First, it may be possible to reduce the number of pads requiredfor accurate identification of target DNA by taking advantage of thisfeature. FIG. 23 shows the potential for this feature in use with theTH01 locus. It predicts that correct identification of all TH01 allelescan be achieved with a set of capture oligos which have five, seven andnine repeat units in combination with the one repeat unit reporter TH01locus. This reduces the required number of analytical test sites fromseven to three. This feature, when combined with the ability to doredundant reporters, could greatly reduce the number of pads requiredfor the analysis of a set of loci for statistically significantgenotyping. Currently this level is approximately 10 loci. Thebeneficial effect of this would be to permit more loci on a single chip,and therefore with larger arrays, the ability to assay multipleindividuals on the same chip thereby reducing the cost of the assay.This would be especially useful for high throughput processing, as willbe required for the STR databasing of felons currently under way.

[0127] Even without a reduction in the number of test sites needed toassay STR alleles, the additional information gained from distinguishinggap mismatch from overlap mismatch will aid in the accuracy of theassay. Any additional information could be incorporated into theultimate statistical analysis of the data to provide an answer which hasa higher probability of being accurate.

Example 11 Determination of STR Allelic Identity of Target DNA byLoopout Analysis

[0128] In another embodiment of STR allele discrimination byhybridization, we have demonstrated that a different oligonucleotidesystem can be used. This method, designated the loopout system, isoutlined in FIG. 9A and 9B, and FIGS. 10A-10G. It is evident from thedrawing that this is an alternative to the sandwich method ofidentifying alleles in a matrix. The loopout system uses an array ofcapture oligos which are distributed in a similar manner to the sandwichformat. The structure of the capture oligos differs from ones in thesandwich format by the presence of locus-specific unique sequenceflanking both ends of the repeat region. Also, the target DNA is labeledand serves as the reporter molecule. The target can be labeled duringamplification by using a PCR primer which is fluorescent (or containsany other suitable molecular adaptation for detection).

[0129] In practice, loopout capture oligos which are specific todifferent alleles of a locus are arrayed in a matrix such thatindividual test sites represent different alleles (See, e.g., FIG. 6).Actual test results are shown in FIG. 24. Labeled target oligo is thenstringently hybridized under electronic conditions to the entire array.The test site with the most stable hybrids will be concordant with theallelic identity of the target DNA. Determining the position of thestable hybrids (and therefore the allele-specific capture oligo attachedto it) identifies the alleles represent in the target DNA. Hybridsformed between capture and reporter oligos which have the same number ofrepeat units are matched. It may also be said that this test site isconcordant with the identity of the target allele. Test sites which arediscordant or contain an unequal number of repeat units between thecapture and the target, will form a hybrid with a loop in either thetarget or capture DNA (See, e.g., FIGS. 10A, 10B and 10D-10G).Discordant sites which have captures with fewer repeat units than thetarget will yield hybrids with loops in the These hybrids will beinherently less stable than the match hybrids. Therefore denaturation byelectronic stringency control will discriminate stable from less stablehybrids indicating the sites of concordancy, and based upon theknowledge of the probes present at those sites, enable the user todetermine the number of repeat units in the target DNA.

Example 12 Detection of Microvariant Allele TH01 9.3

[0130] As STR's become more widely used, deviations within the repeatregions are being discovered with greater frequency. This can betroublesome to conventional size fractionation methods since the marginfor discrimination goes from four bases down to one base. This is in thecase of an insertion or deletion mutations. For transitional ortransversional mutations, the size of the allele doesn't change, eventhough the sequence has become altered.

[0131] Both these classes of mutations, insertion/deletion andtransitional/transversional can be readily detected by our technology.This is primarily due to the fact that Nanogen's approach is ahybridization based assay rather than a sizing method. Thereforecombining terminal nucleotide base stacking with single nucleotidepolymorphism provides a powerful discriminatory tool.

[0132] One well known STR microvariant is the TH01 9.3 allele. It isimportant because it is present in a significant portion of theCaucasian population. The assay for the 9.3 microvariant was essentiallythe same as the normal STR alleles but required special capture andreporter oligo design. The capture oligo contains only three repeatunits (3 ru, FIG. 13). This is because the single base deletion isbetween repeat units 6 and 3 on the target strand. TH01 9.3 target DNAbinding to the capture will be less stable at capture sites containinggreater than three repeat units because there will be a frame-shift inthe repeat region of 9.3. The reporter oligo (Microvar 9.3, FIG. 13) hasbeen designed so that it will bind most stably to the repeat unit regioncontaining the deletion. Additionally, the capture oligo has beendesigned so that target directed base stacking of the capture andreporter DNA will occur only at the 3 ru test site. FIG. 25 shows adetailed sequence alignment of the capture and reporter oligonucleotidesused to detect the TH01 9.3 microvariant. The numbering in the drawingis consistent with that found in FIG. 4C, with the addition of sequence45 which is complementary to the partial repeat unit which constitutesthe microvariant. This particular microvariant reporter has no secondunique flanking sequence 42. This is necessary for analysis of TH01 9.3allele not be a feature of other microvariant-specific reporters. It isevident from this drawing in the hybridization complex which isconcordant for the TH01 9.3 allele, there exists sequencecomplementarity between the target and reporter DNA as well as basestacking between the capture and reporter oligonucleotides. FIG. 26shows the results of an assay of PCR amplified DNA from an individualhomozygote for the TH01 allele.

[0133] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it may be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1 28 1 29 DNA Artificial Sequence Description of Artificial SequenceHuman/ Biotin 1 tcctccctta tttccctcat tcattcatt 29 2 33 DNA ArtificialSequence Description of Artificial Sequence Human/ Biotin 2 tcctcccttatttccctcat tcattcattc att 33 3 37 DNA Artificial Sequence Description ofArtificial Sequence Human/ Biotin 3 tcctccctta tttccctcat tcattcattcattcatt 37 4 41 DNA Artificial Sequence Description of ArtificialSequence Human/ Biotin 4 tcctccctta tttccctcat tcattcattc attcattcat t41 5 45 DNA Artificial Sequence Description of Artificial SequenceHuman/ Biotin 5 tcctccctta tttccctcat tcattcattc attcattcat tcatt 45 649 DNA Artificial Sequence Description of Artificial Sequence Human/Biotin 6 tcctccctta tttccctcat tcattcattc attcattcat tcattcatt 49 7 53DNA Artificial Sequence Description of Artificial Sequence Human/ Biotin7 tcctccctta tttccctcat tcattcattc attcattcat tcattcattc att 53 8 57 DNAArtificial Sequence Description of Artificial Sequence Human/ Biotin 8tcctccctta tttccctcat tcattcattc attcattcat tcattcattc attcatt 57 9 10DNA Artificial Sequence Description of Artificial Sequence Human/ BodipyTexas Red 9 cattcaccat 10 10 11 DNA Artificial Sequence Description ofArtificial Sequence Human/ Bodipy Texas Red 10 cattcattca c 11 11 10 DNAArtificial Sequence Description of Artificial Sequence Human/ BodipyTexas Red 11 caccatggag 10 12 10 DNA Artificial Sequence Description ofArtificial Sequence Human/ Bodipy Texas Red 12 catcattcat 10 13 54 DNAHomo sapiens 13 acacagactc catggtgaat gaatgaatga atgaatgagg gaaataagggagga 54 14 58 DNA Homo sapiens 14 acacagactc catggtgaat gaatgaatgaatgaatgaat gagggaaata agggagga 58 15 62 DNA Homo sapiens 15 acacagactccatggtgaat gaatgaatga atgaatgaat gaatgaggga aataagggag 60 ga 62 16 66DNA Homo sapiens 16 acacagactc catggtgaat gaatgaatga atgaatgaatgaatgaatga gggaaataag 60 ggagga 66 17 70 DNA Homo sapiens 17 acacagactccatggtgaat gaatgaatga atgaatgaat gaatgaatga atgagggaaa 60 taagggagga 7018 73 DNA Homo sapiens 18 acacagactc catggtgaat gaatgaatga atgaatgaatgatgaatgaa tgaatgaggg 60 aaataaggga gga 73 19 74 DNA Homo sapiens 19acacagactc catggtgaat gaatgaatga atgaatgaat gaatgaatga atgaatgagg 60gaaataaggg agga 74 20 78 DNA Homo sapiens 20 acacagactc catggtgaatgaatgaatga atgaatgaat gaatgaatga atgaatgaat 60 gagggaaata agggagga 78 2141 DNA Artificial Sequence Description of Artificial Sequence Human/Biotin 21 ttagggaacc ctcactgaat gaatgaatga atgaatgaat g 41 22 10 DNAArtificial Sequence Description of Artificial Sequence Human/ BodipyTexas Red 22 aatgtttggg 10 23 11 DNA Artificial Sequence Description ofArtificial Sequence Human/ Bodipy Texas Red 23 aatgaatgtt t 11 24 10 DNAArtificial Sequence Description of Artificial Sequence Human/ BodipyTexas Red 24 tttgggcaaa 10 25 53 DNA Homo sapiens 25 tttgcccaaacattcattca ttcattcatt cattcagtga gggttcccta agt 53 26 48 DNA Homosapiens 26 gccttcatag atagaagata gatagattag atagatagat agatagat 48 27 10DNA Homo sapiens 27 agataggaag 10 28 69 DNA Homo sapiens 28 tgttctaagtacttcctatc tatctatcta tctatctatc taatctatct atcttctatc 60 tatgaaggc 69

We claim:
 1. A method for determining the nature of a genetic variant ina nucleic acid target, comprising the steps of: providing a plurality ofhybridization complex assays on an electronic test device, where thehybridization complex comprises: a nucleic acid target containing agenetic variant nucleic acid sequence, a capture probe having a selectedsequence complementary to a first target sequence, and a reporter probehaving a selected sequence complementary to a different portion of thesame target strand, wherein the reporter probe and capture probe form ahybridization complex with the target such that the termini of thecapture and reporter are juxtaposed, said capture probe and reporterprobe being selected such that the base-stacking between the juxtaposedtermini is disrupted within a discordant complex, and wherein theelectronic test device subjects one or more of the assay components toan electric field.
 2. The method of claim 1 for determining the natureof a genetic variant wherein the electronic test device includes aplurality of test sites.
 3. The method of claim 2 for determining thenature of a genetic variant wherein a test site include amicroelectrode.
 4. The method of claim 3 for determining the nature of agenetic variant wherein a test site includes a permeation layer disposedadjacent the microelectrode.
 5. The method of claim 1 for determiningthe nature of a genetic variant wherein two different hybridizationcomplexes are distinguished at a single site with distinguishablereporters.
 6. The method of claim 1 for determining the nature of agenetic variant wherein two different hybridization complexes aredistinguished at multiple sites with different reporters.
 7. A kit forperforming the method of claim 1 for determining the nature of a geneticvariant including said reporter probe and said capture probe.
 8. Themethod of claim 1 for determining the nature of a genetic variantwherein the variant constitutes the number of repetitive DNA sequences.9. The method of claim 1 wherein the genetic variants include deletions.10. The method of claim 1 wherein the genetic variants includeinsertions.
 11. The method of claim 1 wherein the genetic variantsinclude transitions.
 12. The method of claim 1 wherein the geneticvariants include transversions.
 13. The method of claim 1 wherein thegenetic variants include single nucleotide variants.
 14. The method ofclaim 1 wherein the genetic variants include mutations.
 15. The methodof claim 1 wherein the genetic variants include point mutations.
 16. Themethod of claim 1 wherein the genetic variant affects a single base. 17.The method of claim 5 wherein the genetic variant affects more than asingle base.
 18. The method of claim 1 wherein multiple test locationsindicate the nature of the target sequence by the detection of geneticvariants in the target sequence.
 19. The method of claim 1 whereindistinguishable reporters at a single test site location indicate thenature of the target sequence by the detection of genetic variants inthe target sequence.
 20. The method of claim 1 wherein the plurality ofhybridization complex assays form an array.
 21. The method of claim 20wherein the location of the test site in the array indicates the natureof the genetic variant.
 22. The method of claim 1 wherein a test site isdetermined to be concordant when the hybridization complex present thereis stable relative to other test sites for the same locus.
 23. Themethod of claim 1 wherein the stability is enhanced by complementarymatch of bases in the hybridization complex including the capture,reporter and target.
 24. The method of claim 1 wherein the stability isenhanced by juxtaposed terminal nucleotides of the reporter and capturebeing contiguous to permit base stacking.
 25. The method of claim 1wherein the hybridization complex assay is determined to be concordantwhen a distinguishable reporter present there is stable relative toother distinguishable reporters for the same locus.
 26. The method ofclaim 1 wherein the base stacked pair is 5′GpC3′.
 27. The method ofclaim 1 wherein the base stacked pair is 5′TpA3′.
 28. The method ofclaim 1 further including modifications of terminal nucleotides whichincrease base stacking.
 29. The method of claim 1 wherein stability isenhanced with ligation techniques.
 30. The method of claim 1 whereindiscordance includes a genetic variant.
 31. The method of claim 30wherein the genetic variant is in the target sequence.
 32. The method ofclaim 31 wherein the genetic variant in the target sequence is selectedfrom the group comprising: insertions, deletions, transitions andtransversions.
 33. The method of claim 1 wherein discordance includesgenetic variations which are greater than a single nucleotide.
 34. Themethod of claim 1 wherein the number of loci analyzed at one time isone.
 35. The method of claim 1 wherein the number of loci analyzed atone time is three.
 36. The method of claim 1 wherein the number of locianalyzed at one time is less than five.
 37. The method of claim 1wherein the number of loci analyzed at one time is less than ten. 38.The method of claim 1 wherein the number of loci analyzed at one time isgreater than ten.
 39. The method of claim 1 wherein the number of locianalyzed at one time is less than one hundred.
 40. The method of claim 1wherein the number of loci analyzed at one time is greater than onehundred.
 41. The method of claim 1 wherein the disruption of thejuxtaposed terminii results in destabilization of the hybridizationcomplex.
 42. The method of claim 1 wherein the hybridization stabilityis determined, at least in part, by electronic stringency (ESC) control.43. The method of claim 1 wherein the hybridization stability isdetermined, at least in part, by thermal regulation of stringency. 44.The method of claim 1 wherein the hybridization stability is determined,at least in part, by chemical regulation of stringency.
 45. The methodof claim 1 wherein the hybridization stability is determined, at leastin part, by electronic stringency (ESC) and thermal control.
 46. Themethod of claim 1 wherein the hybridization stability is determined, atleast in part, by electronic stringency (ESC) and chemical control. 47.The method of claim 1 wherein the hybridization stability is determined,at least in part, by electronic stringency (ESC), thermal and chemicalcontrol.
 48. The method of claim 1 wherein the electric field controlincludes electronic addressing of the target.
 49. The method of claim 1wherein the electric field control includes electronic addressing of thecapture.
 50. The method of claim 1 wherein the electric field controlincludes electronic addressing of the reporter.
 51. The method of claim1 wherein the electric field control includes electronic control ofhybridizing the target in the complex.
 52. The method of claim 1 whereinthe electric field control includes electronic control of hybridizingthe capture in the complex.
 53. The method of claim 1 wherein theelectric field control includes electronic control of hybridizing thereporter in the complex.
 54. The method of claim 1 wherein the electricfield control includes electronic control of formation of thehybridization complex.
 55. The method of claim 1 further includingelectronic stringency conditions during the hybridizations of captureprobe with the nucleic acid target.
 56. The method of claim 55 whereininitial hybridization step occurs in 10 minutes or less.
 57. The methodof claim 55 wherein initial hybridization step occurs in 5 minutes orless.
 58. The method of claim 55 wherein initial hybridization stepoccurs in 1 minute or less.
 59. The method of claim 1 further includingelectronic stringency conditions during the hybridization of thereporter probe with the capture probe nucleic acid target hybridizationcomplex.
 60. The method of claim 1 wherein the hybridization complex islabeled.
 61. The method of claim 60 further including the step ofdetecting the amounts of labeled hybridization complex at the testsites.
 62. The method of claim 61 wherein the detecting is imaging. 63.The method of claim 62 wherein the imaging is optical imaging.
 64. Themethod of claim 62 wherein the imaging is electronic imaging.
 65. Themethod of claim 62 wherein the imaging is CCD imaging.
 66. The method ofclaim 62 wherein the imaging is integrated optical imaging.
 67. Themethod of claim 62 wherein the imaging detection is quantified.
 68. Themethod of claim 1 further including a statistical analysis step.
 69. Themethod of claim 60 wherein the labeled portion of complex is the target.70. The method of claim 60 wherein the labeled portion of complex is thecapture.
 71. The method of claim 60 wherein the labeled portion ofcomplex is the reporter.
 72. The method of claim 60 wherein the labelingis by fluorescent labeling.
 73. The method of claim 72 wherein thefluorescent labeling is with Bodipy Texas Red.
 74. The method of claim72 wherein the fluorescent labeling is with Bodipy 630/650.
 75. Themethod of claim 72 wherein the fluorescent labeling is with LuciferYellow.
 76. The method of claim 60 wherein the labeling is bycolormetric labeling.
 77. The method of claim 60 wherein the labeling isby chemiluminescent labeling.
 78. The method of claim 1 furtherincluding energy transfer between molecules in the hybridizationcomplex.
 79. The method of claim 1 further including fluorescentperturbation analysis.
 80. The method of claim 78 wherein the energytransfer includes quenching.
 81. The method of claim 1 further includinga redundant assay.
 82. The method of claim 81 further including the stepof repeating the redundant assay until a statistically significantresult is obtained.
 83. The method of claim 81 wherein the redundantassay includes multiple arrays.
 84. The method of claim 1 wherein thetarget DNA is purified.
 85. The method of claim 1 wherein the target isunamplified.
 86. The method of claim 1 wherein the target is amplified.87. The method of claim 1 wherein the target is applied to a reductionof test sites necessary to identify the number of repeat units in thetarget DNA.
 88. The method of claim 87 wherein the reduction increasesthe statistical significance of results.
 89. The method of claim 1wherein the target material constitutes homozygous allele for a locus.90. The method of claim 1 wherein the target material constitutesheterozygous allele for a locus.
 91. The method of claim 1 wherein thetarget material constitutes more than one allele per locus for a mixedsample.
 92. The method of claim 91 wherein the mixed sample furtherincludes sample from more than one individual.
 93. The method of claim92 wherein the mixed sample further includes tumor tissue mixed withnormal tissue.
 94. A method for determining the nature of a geneticvariant in a nucleic acid target, comprising the steps of: providing aplurality of hybridization complex assays on a test device, where thehybridization complex comprises: a nucleic acid target containing agenetic variant nucleic acid sequence, a capture probe having a selectedsequence complementary to a first target sequence, and a reporter probehaving a selected sequence complementary to a different portion of thesame target strand, wherein the reporter probe and capture probe form ahybridization complex with the target such that the termini of thecapture and reporter are juxtaposed, and selecting the capture probe andreporter probe from a plurality of options by varying at least theposition of the juxtaposed termini relative to the genetic variant, suchthat the base-stacking stabilization between the juxtaposed termini isdetermined.
 95. The method of claim 94 such that the base-stackingstabilization between the juxtaposed termini is maximized within aconcordant hybridization complex.
 96. The method of claim 94 such thatthe base-stacking stabilization between the juxtaposed termini isminimized within a discordant hybridization complex.
 97. The method ofclaim 94 such that the differential base-stacking stabilization betweenthe juxtaposed termini is maximized for a discordant and concordanthybridization complex.
 98. The method of claim 94 wherein the geneticvariant is immediately adjacent the termini.
 99. The method of claim 94wherein the genetic variant is one base removed from the termini. 100.The method of claim 94 wherein the genetic variant is two or more basesremoved from the termini.
 101. A kit for performing the method of claim94 including said reporter probe and said capture probe.
 102. The methodof claim 94 wherein the base-stacking is maximized by selection of theterminal bases.
 103. The method of claim 94 wherein the base-stacking ismaximized by selection of the position of the genetic variant relativeto the juxtaposed termini.
 104. The method of claim 94 wherein thebase-stacking is maximized by selection of complementary sequences. 105.The method of claim 94 wherein the base-stacking is maximized byselection of the terminal bases and complementary sequences.
 106. Themethod of claim 94 wherein the base-stacking is maximized by selectionof the terminal bases, complementary sequences and position of thegenetic variant relative to the juxtaposed termini.
 107. The method ofclaim 94 for determining the nature of a genetic variant wherein thetest device includes a plurality of test sites.
 108. The method of claim107 for determining the nature of a genetic variant wherein a test siteincludes a permeation layer disposed adjacent the microelectrode. 109.The method of claim 94 for determining the nature of a genetic variantwherein two different hybridization complexes are distinguished at asingle site with distinguishable reporters.
 110. The method of claim 94for determining the nature of a genetic variant wherein two differenthybridization complexes are distinguished at multiple sites withdifferent reporters.
 111. A kit for performing the method of claim 94for determining the nature of a genetic variant including said reporterprobe and said capture probe.
 112. The method of claim 94 fordetermining the nature of a genetic variant wherein the variantconstitutes the number of repetitive DNA sequences.
 113. The method ofclaim 94 wherein the genetic variants include deletions.
 114. The methodof claim 94 wherein the genetic variants include insertions.
 115. Themethod of claim 94 wherein the genetic variants include transitions.116. The method of claim 94 wherein the genetic variants includetransversions.
 117. The method of claim 94 wherein the genetic variantsinclude single nucleotide variants.
 118. The method of claim 94 whereinthe genetic variants include mutations.
 119. The method of claim 94wherein the genetic variants include point mutations.
 120. The method ofclaim 94 wherein the genetic variant affects a single base.
 121. Themethod of claim 119 wherein the genetic variant affects more than asingle base.
 122. The method of claim 94 wherein multiple test locationsindicate the nature of the target sequence by the detection of geneticvariants in the target sequence.
 123. The method of claim 94 whereindistinguishable reporters at a single test site location indicate thenature of the target sequence by the detection of genetic variants inthe target sequence.
 124. The method of claim 94 wherein the pluralityof hybridization complex assays form an array.
 125. The method of claim124 wherein the location of the test site in the array indicates thenature of the genetic variant.
 126. The method of claim 94 wherein atest site is determined to be concordant when the hybridization complexpresent there is stable relative to other test sites for the same locus.127. The method of claim 94 wherein the stability is enhanced bycomplementary match of bases in the hybridization complex including thecapture, reporter and target.
 128. The method of claim 94 wherein thehybridization complex assay is determined to be concordant when adistinguishable reporter present there is stable relative to otherdistinguishable reporters for the same locus.
 129. The method of claim94 wherein the base stacked pair is 5′GpC3′.
 130. The method of claim 94wherein the base stacked pair is 5′TpA3′.
 131. The method of claim 94further including modifications of terminal nucleotides which increasebase stacking.
 132. The method of claim 94 wherein stability is enhancedwith ligation techniques.
 133. The method of claim 94 whereindiscordance includes a genetic variant.
 134. The method of claim 133wherein the genetic variant is in the target sequence.
 135. The methodof claim 133 wherein the genetic variant in the target sequence isselected from the group comprising: insertions, deletions, transitionsand transversions.
 136. The method of claim 94 wherein discordanceincludes genetic variations which are greater than a single nucleotide.137. The method of claim 94 wherein the number of loci analyzed at onetime is one.
 138. The method of claim 94 wherein the number of locianalyzed at one time is three.
 139. The method of claim 94 wherein thenumber of loci analyzed at one time is less than five.
 140. The methodof claim 94 wherein the number of loci analyzed at one time is less thanten.
 141. The method of claim 94 wherein the number of loci analyzed atone time is greater than ten.
 142. The method of claim 94 wherein thenumber of loci analyzed at one time is less than one hundred.
 143. Themethod of claim 94 wherein the number of loci analyzed at one time isgreater than one hundred.
 144. The method of claim 94 wherein thedisruption of the juxtaposed termini results in destabilization of thehybridization complex.
 145. The method of claim 94 wherein thedisruption of complementary sequences results in destabilization of thehybridization complex.
 146. The method of claim 94 wherein thedisruption of the juxtaposed termini and complementary sequences resultsin destabilization of the hybridization complex.
 147. The method ofclaim 94 wherein the hybridization stability is determined, at least inpart, by thermal regulation of stringency.
 148. The method of claim 94wherein the hybridization stability is determined, at least in part, bychemical regulation of stringency.
 149. The method of claim 94 whereininitial hybridization step occurs in 10 minutes or less.
 150. The methodof claim 94 wherein initial hybridization step occurs in 5 minutes orless.
 151. The method of claim 94 wherein initial hybridization stepoccurs in 1 minute or less.
 152. The method of claim 94 wherein thehybridization complex is labeled.
 153. The method of claim 152 furtherincluding the step of detecting the amounts of labeled hybridizationcomplex at the test sites.
 154. The method of claim 153 wherein thedetecting is imaging.
 155. The method of claim 154 wherein the imagingis optical imaging.
 156. The method of claim 154 wherein the imaging iselectronic imaging.
 157. The method of claim 154 wherein the imaging isCCD imaging.
 158. The method of claim 154 wherein the imaging isintegrated optical imaging.
 159. The method of claim 154 wherein theimaging detection is quantified.
 160. The method of claim 94 furtherincluding a statistical analysis step.
 161. The method of claim 94wherein the labeled portion of complex is the target.
 162. The method ofclaim 94 wherein the labeled portion of complex is the capture.
 163. Themethod of claim 94 wherein the labeled portion of complex is thereporter.
 164. The method of claim 94 wherein the labeling is byfluorescent labeling.
 165. The met hod of claim 164 wherein thefluorescent labeling is with Bodipy Texas Red.
 166. The method of claim164 wherein the fluorescent labeling is with Bodipy 630/650.
 167. Themethod of claim 164 wherein the fluorescent labeling is with LuciferYellow.
 168. The method of claim 152 wherein the labeling is bycolormetric labeling.
 169. The method of claim 152 wherein the labelingis by chemiluminescent labeling.
 170. The method of claim 94 furtherincluding energy transfer between molecules in the hybridizationcomplex.
 171. The method of claim 170 wherein the energy transferincludes quenching.
 172. The method of claim 94 further including aredundant assay.
 173. The method of claim 172 further including the stepof repeating the redundant assay until a statistically significantresult is obtained.
 174. The method of claim 172 wherein the redundantassay includes multiple arrays.
 175. The method of claim 94 wherein thetarget DNA is purified.
 176. The method of claim 94 wherein the targetis unamplified.
 177. The method of claim 94 wherein the target isamplified.
 178. The method of claim 94 wherein the target is applied toa reduction of test sites necessary to identify the number of repeatunits in the target DNA.
 179. The method of claim 178 wherein thereduction increases the statistical significance of results.
 180. Themethod of claim 94 wherein the target material constitutes homozygousallele for a locus.
 181. The method of claim 94 wherein the targetmaterial constitutes heterozygous allele for a locus.
 182. The method ofclaim 94 wherein the target material constitutes more than one alleleper locus for a mixed sample.
 183. The method of claim 182 wherein themixed sample further includes sample from more than one individual. 184.The method of claim 183 wherein the mixed sample further includes tumortissue mixed with normal tissue.
 185. A method for determining thenature of a genetic variant in a nucleic acid target, comprising thesteps of: providing a plurality of hybridization complex assays at aplurality of locations on an electronic test device, where thehybridization complex comprises: a nucleic acid target containing agenetic variant nucleic acid sequence, a capture probe having a selectedsequence complementary to a first target sequence, and a reporter probehaving a selected sequence complementary to a different portion of thesame target strand, wherein at least two sites contain target fromdifferent sources, and wherein the reporter probe and capture probe forma hybridization complex with the target such that the termini of thecapture and reporter are juxtaposed.
 186. The method of claim 185wherein the capture probe and reporter probe are selected such that thebase-stacking between the juxtaposed termini which is disrupted within adiscordant complex.
 187. The method of claim 185 wherein the geneticvariant is immediately adjacent the termini.
 188. The method of claim185 wherein the genetic variant is one base removed from the termini.189. The method of claim 184 wherein the genetic variant is two or morebases removed from the termini.
 190. A method for uniquely identifyingany of N alleles in a genetic sample comprising the steps of: performingM tests on the genetic sample, where M is less than N, wherein each testdetects concordance, discordance, and each of at least two degrees ofdiscordance, and combining the data from the M tests to uniquelyidentify the alleles.
 191. The method of claim 190 wherein the selectionof the range of tests spans the set of N alleles.